Ligate the PCR product into a plasmid and transform bacteria. Students directly clone their PCR products into the pJET1.2 vector and immediately transform bacteria using a protocol that takes less than 2 hours to go from purified PCR product to transformed bacteria plated on agar.

Students first remove the 3'-dA overhang from their PCR product which results from amplification with Taq polymerase using a proofreading polymerase. A 10 minute protocol is then used to ligate the blunted PCR product into a pre-opened and blunted vector — pJET1.2. The pJET1.2 plasmid positively selects for successful insertion of fragments because the cloned fragment inserts into a lethal gene present in the plasmid that prevents its activation. Bacteria that are transformed with re-ligated vector activate the gene and are killed. This results in higher transformation efficiency than traditional blue-white cloning. Bgl II restriction sites are located on either side of the pJET1.2 cloning site, which allow students to determine if their gene of interest was successfully ligated once they have isolated candidate plasmids.

To transform the ligation into bacteria, students make competent cells by inoculating specialized growth media and performing a series of microcentrifugations and washes in a specialized transformation buffer. Competent cells are then added to the ligations on ice and plated directly on warm agar plates. Between 10 and 100 colonies should be expected from transformed ligations with the Cloning and Sequencing Explorer Series.

The ligation and transformation module is an integral component of the Cloning and Sequencing Explorer Series. Use this module to directly ligate PCR products into the pJET1.2 plasmid vector and immediately transform freshly prepared competent bacteria with the ligation reaction. Competent cells are then plated directly on warm agar plates and incubated overnight at 37°C.

Once transformed bacteria have been propagated, plasmid DNA can be isolated and digested with BglII enzyme. The BglII restriction sites located on either side of the pJET1.2 cloning site allow students to determine if their PCR product was successfully ligated.

This module may also be purchased separately and used to clone any PCR product. The entire ligation and transformation protocol takes approximately 2 hours to complete and does not require commercial competent cells, a refrigerated microcentrifuge, or a –80°C freezer, making it the method of choice for educators worldwide.

What is the education discount policy?

For more than 15 years, Bio-Rad has made science education a major priority. To support this effort, the company has implemented a discount policy that allows high school and college teaching laboratories to purchase kits, instruments, reagents, and other equipment at preferred prices.

How do I apply for the education discount?

If you are an educator at the high school or college level, visit our Education Discount Policy page to establish an education account number.

If you are placing an order, you may proceed with your order; the account price will be applied if it is lower than the list price. If the problem persists, please call customer service at
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